Analysis of the expression level and predictive value of CLEC16A|miR-654-5p|RARA regulatory axis in the peripheral blood of patients with ischemic stroke based on biosignature analysis.

Introduction: Ischemic stroke (IS) is a cerebrovascular disease that can be disabling and fatal, and there are limitations in the clinical treatment and prognosis of IS. It has been reported that changes in the expression profile of circRNAs have been found during injury in ischemic stroke, and circRNAs play an important role in the IS cascade response. However, the specific mechanisms involved in the pathogenesis of IS are not yet fully understood, and thus in-depth studies are needed. Methods: In this study, one circRNA dataset (GSE161913), one miRNA dataset (GSE60319) and one mRNA dataset (GSE180470) were retrieved from the Gene Expression Omnibus (GEO) database and included, and the datasets were differentially expressed analyzed by GEO2R and easyGEO to get the DEcircRNA, DEmiRNA and DEmRNA, and DEmRNA was enriched using ImageGP, binding sites were predicted in the ENCORI database, respectively, and the competitive endogenous RNA (ceRNA) regulatory network was visualized by the cytoscape software, and then selected by MCC scoring in the cytoHubba plugin Hub genes. In addition, this study conducted a case-control study in which blood samples were collected from stroke patients and healthy medical examiners to validate the core network of ceRNAs constructed by biosignature analysis by real-time fluorescence quantitative qRT-PCR experiments. Results: A total of 233 DEcircRNAs, 132 DEmiRNAs and 72 DEmRNAs were screened by bioinformatics analysis. circRNA-mediated ceRNA regulatory network was constructed, including 148 circRNAs, 43 miRNAs and 44 mRNAs. Finally, CLEC16A|miR-654-5p|RARA competitive endogenous regulatory axis was selected for validation by qRT-PCR, and the validation results were consistent with the bioinformatics analysis. Discussion: In conclusion, the present study establishes a new axis of regulation associated with IS, providing new insights into the pathogenesis of IS.

Enhancement of Repeat-Mediated Deletion Rearrangement Induced by Particle Irradiation in a RecA-Dependent Manner in Escherichia coli.

Repeat-mediated deletion (RMD) rearrangement is a major source of genome instability and can be deleterious to the organism, whereby the intervening sequence between two repeats is deleted along with one of the repeats. RMD rearrangement is likely induced by DNA double-strand breaks (DSBs); however, it is unclear how the complexity of DSBs influences RMD rearrangement. Here, a transgenic Escherichia coli strain K12 MG1655 with a lacI repeat-controlled amp activation was used while taking advantage of particle irradiation, such as proton and carbon irradiation, to generate different complexities of DSBs. Our research confirmed the enhancement of RMD under proton and carbon irradiation and revealed a positive correlation between RMD enhancement and LET. In addition, RMD enhancement could be suppressed by an intermolecular homologous sequence, which was regulated by its composition and length. Meanwhile, RMD enhancement was significantly stimulated by exogenous λ-Red recombinase. Further results investigating its mechanisms showed that the enhancement of RMD, induced by particle irradiation, occurred in a RecA-dependent manner. Our finding has a significant impact on the understanding of RMD rearrangement and provides some clues for elucidating the repair process and possible outcomes of complex DNA damage.

From Photon Beam to Accelerated Particle Beam: Antimetastasis Effect of Combining Radiotherapy With Immunotherapy.

Cancer is one of the major diseases that seriously threaten the human health. Radiotherapy is a common treatment for cancer. It is noninvasive and retains the functions of the organ where the tumor is located. Radiotherapy includes photon beam radiotherapy, which uses X-rays or gamma rays, and particle beam radiotherapy, using beams of protons and heavy ions. Compared with photon beam radiotherapy, particle beam radiotherapy has excellent dose distribution, which enables it to kill the primary tumor cells more effectively and simultaneously minimize the radiation-induced damage to normal tissues and organs surrounding the tumor. Despite the excellent therapeutic effect of particle beam radiotherapy on the irradiated tumors, it is not an effective treatment for metastatic cancers. Therefore, developing novel and effective treatment strategies for cancer is urgently needed to save patients with distant cancer metastasis. Immunotherapy enhances the body's own immune system to fight cancer by activating the immune cells, and consequently, to achieve the systemic anticancer effects, and it is considered to be an adjuvant therapy that can enhance the efficacy of particle beam radiotherapy. This review highlights the research progress of the antimetastasis effect and the mechanism of the photon beam or particle beam radiotherapy combined with immunotherapy and predicts the development prospects of this research area.

Th Balance-Related Host Genetic Background Affects the Therapeutic Effects of Combining Carbon-Ion Radiation Therapy With Dendritic Cell Immunotherapy.

PURPOSE: The goal of this study is to clarify the underlying mechanisms of metastasis suppression by carbon-ion radiotherapy combined with immature dendritic cell immunotherapy (CiDC), which was shown previously to suppress pulmonary metastasis in an NR-S1-bearing C3H/He mouse model. METHODS AND MATERIALS: Mouse carcinoma cell lines (LLC, LM8, Colon-26, and Colon-26MGS) were grafted into the right hind paw of syngeneic mice (C57BL/6J, C3H/He, and BALB/c). Seven days later, the tumors on the mice were locally irradiated with carbon ions (290 MeV/n, 6 cm spread-out Bragg peak, 1 or 2 Gy). At 1.5 days after irradiation, bone marrow-derived immature dendritic cells (iDCs) were administrated intravenously into a subset of the mice. The number of lung metastases was evaluated within 3 weeks after irradiation. In vitro-cultured cancer cells were irradiated with carbon ions (290 MeV/n, mono-energy, LET approximately 70-80 keV/µm), and then cocultured with iDCs for 3 days to determine the DC maturation. RESULTS: CiDC effectively repressed distant lung metastases in cancer cell (LLC and LM8)-bearing C57BL/6J and C3H/He mouse models. However, Colon-26- and Colon-26MGS-bearing BALB/c models did not show enhancement of metastasis suppression by combination treatment. This result was evaluated further by comparing LM8-bearing C3H/He and LLC-bearing C57BL/6J models with a Colon-26-bearing BALB/c model. In vitro coculture assays demonstrated that all irradiated cell lines were able to activate C3H/He- or C57BL/6J-derived iDCs into mature DCs, but not BALB/c-derived iDCs. CONCLUSIONS: The genetic background of the host could have a strong effect on the potency of combination therapy. Future animal and clinical testing should evaluate host genetic factors when evaluating treatment efficacy.

Combined Effects of Proton Radiation and Simulated Microgravity on the Cell Viability and ALP Activity of Murine Osteoblast Cells.

Proton radiation (PR) and microgravity (µG) are two key factors that impact living things in space. This study aimed to explore the combined effects of PR and simulated µG (SµG) on bone function. Mouse embryo osteoblast precursor cells (MC3T3-E1) were irradiated with proton beams and immediately treated with SµG for 2 days using a three-dimensional clinostat. All samples were subjected to cell viability, alkaline phosphatase (ALP) activity and transcriptome assays. The results showed that cell viability decreased with increasing doses of PR. The peak ALP activity after PR or SµG alone was lower than that obtained with the non-treatment control. No difference in cell viability or ALP activity was found between 1 Gy PR combined with SµG (PR-SµG) and PR alone. However, 4 Gy PR-SµG resulted in decreased cell viability and ALP activity compared with those obtained with PR alone. Furthermore, Gene Ontology analysis revealed the same trend. These results revealed that PR-SµG may lead to reductions in the proliferation and differentiation capacities of cells in a dose-dependent manner. Our data provide new insights into bone-related hazards caused by multiple factors, such as PR and µG, in the space environment.

From Classical Radiation to Modern Radiation: Past, Present, and Future of Radiation Mutation Breeding.

Radiation mutation breeding has been used for nearly 100 years and has successfully improved crops by increasing genetic variation. Global food production is facing a series of challenges, such as rapid population growth, environmental pollution and climate change. How to feed the world's enormous human population poses great challenges to breeders. Although advanced technologies, such as gene editing, have provided effective ways to breed varieties, by editing a single or multiple specific target genes, enhancing germplasm diversity through mutation is still indispensable in modern and classical radiation breeding because it is more likely to produce random mutations in the whole genome. In this short review, the current status of classical radiation, accelerated particle and space radiation mutation breeding is discussed, and the molecular mechanisms of radiation-induced mutation are demonstrated. This review also looks into the future development of radiation mutation breeding, hoping to deepen our understanding and provide new vitality for the further development of radiation mutation breeding.

Characterization of a Novel Murine Colon Carcinoma Subline with High-Metastatic Activity Established by In Vivo Selection Method.

The establishment of cancer cell lines, which have different metastatic abilities compared with the parental cell, is considered as an effective approach to investigate mechanisms of metastasis. A highly metastatic potential mouse colon cancer cell subline, Colon-26MGS, was derived from the parental cell line Colon-26 by in vivo selection using continuous subcutaneous implanting to immunocompetent mice. To clarify the mechanisms involved in the enhancement of metastasis, morphological characteristics, cell proliferation, and gene expression profiles were compared between Colon-26MGS and the parental cell. Colon-26MGS showed over 10 times higher metastatic ability compared with the parental cell, but there were no differences in morphological characteristics and in vitro proliferation rates. In addition, the Colon-26MGS-bearing mice exhibited no marked change of splenocyte population and lung pre-metastatic niche with tumor-free mice, but there were significant differences compared to Colon-26-bearing mice. RNA-seq analyses indicated that immune costimulatory molecules were significantly up-regulated in Colon-26MGS. These results suggest that Colon-26MGS showed not only higher metastatic activity, but also less induction property of host immune response compared to parental Colon-26. Colon-26MGS has proven to be a novel useful tool for studying multiple mechanisms involving metastasis enhancement.

Anti-Metastatic Benefits Produced by Hyperthermia and a CCL3 Derivative.

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 105 cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 µg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated after 18 days was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

Temporary Loading Prevents Cancer Progression and Immune Organ Atrophy Induced by Hind-Limb Unloading in Mice.

Although the body's immune system is altered during spaceflight, the effects of microgravity (µG) on tumor growth and carcinogenesis are, as yet, unknown. To assess tumor proliferation and its effects on the immune system, we used a hind-limb unloading (HU) murine model to simulate µG during spaceflight. HU mice demonstrated significantly increased tumor growth, metastasis to the lung, and greater splenic and thymic atrophy compared with mice in constant orthostatic suspension and standard housing controls. In addition, mice undergoing temporary loading during HU (2 h per day) demonstrated no difference in cancer progression and immune organ atrophy compared with controls. Our findings suggest that temporary loading can prevent cancer progression and immune organ atrophy induced by HU. Further space experiment studies are warranted to elucidate the precise effects of µG on systemic immunity and cancer progression.

LET dependence on killing effect and mutagenicity in the model filamentous fungus Neurospora crassa.

PURPOSE: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora. MATERIALS AND METHODS: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined. RESULTS: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET). CONCLUSIONS: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.

Intravenous dendritic cell administration enhances suppression of lung metastasis induced by carbon-ion irradiation.

Carbon-ion radiotherapy (CIRT) is an advanced radiotherapy and has achieved good local control, even in tumors that are resistant to conventional photon beam radiotherapy (PBRT). However, distant metastasis control is an important issue. Recently, the combination of radiotherapy and immunotherapy has attracted the attention. In immunotherapy, dendritic cells (DCs) play a pivotal role in the anti-tumor immune system. However, the mechanisms underlying the combination therapy of DCs and radiotherapy have been unclear. In the present study, we evaluated anti-metastatic effects of this combination therapy, focused on the irradiation type and the route of DC administration, using a mouse model. C3H/He mice bearing NR-S1 cells were treated with CIRT or PBRT, using biologically equivalent doses. Subsequently, DCs were administered intratumorally (IT) or intravenously (IV). IV and IT DC administrations combined with CIRT to the local tumor, but not alone, significantly suppressed pulmonary metastasis, whereas the combination of DCs with PBRT suppressed metastasis at a relatively higher dose. Additionally, the anti-metastatic effect was greater in IV DC administration compared with in IT DC administration in both CIRT and PBRT. The expression levels of CD40 and IL-12 in DCs were significantly increased after co-culturing with CIRT-treated NR-S1 cells. In addition, IV administration of those co-cultured DCs significantly suppressed pulmonary metastasis. Furthermore, ecto-calreticulin levels from CIRT-treated NR-S1 cells significantly increased compared with those of a PBRT-treated tumor. Taken together, these results suggest that local CIRT combined with IV DCs augments an immunogenicity of the tumor cells by ecto-calreticulin expression and the maturation of DCs to stimulate anti-tumor immunity to decrease lung metastases.

The Future of Combining Carbon-Ion Radiotherapy with Immunotherapy: Evidence and Progress in Mouse Models.

After >60 years since the first treatment, particle radiation therapy (RT) is now used to treat various types of tumors worldwide. Particle RT results in favorable outcomes, especially in local control, because of its biological properties and excellent dose distribution. However, similar to other types of cancer treatment, metastasis control is a crucial issue. Notably, immunotherapy is used for cancer treatment with high risk for recurrence and/or metastasis. These 2 cancer therapies could be ideal, complementary partners for noninvasive cancer treatment. In this review, we will focus on preclinical studies combining particle RT, especially carbon ion RT, and immunotherapy.

The type of mutations induced by carbon-ion-beam irradiation of the filamentous fungus Neurospora crassa.

Heavy-ion beams are known to cause great damage to cellular components and are particularly renowned for their ability to generate DNA double-strand breaks (DSBs). To gain insight into the mutagenic effect of carbon-ion beams and how such damage is repaired by the cell, Neurospora crassa mutants deficient in one of three components involved in the repair of DSBs, nonhomologous end-joining (NHEJ), homologous recombination repair (HR), and the Mre11-Rad50-Xrs2 (MRX) complex, were irradiated with a carbon-ion beam and killing effect, mutation frequencies, and the type of mutation incurred by survivors were analysed. The sensitivity of the NHEJ-deficient strain (mus-52) was higher than that of the wild-type and the HR-deficient (mei-3) strains at low doses of radiation, but was little changed as the level increased. As a result both the wild-type and HR-deficient strains were more sensitive than the NHEJ-deficient strain at high radiation levels. In addition, the frequency of forward mutation at the adenine-3 (ad-3) loci of the NHEJ-deficient mutant was lower than that of the wild-type strain at all levels, while the mutation frequency of the HR-deficient strain was consistently ∼3-fold higher than the wild-type. From the comparison of mutation type of each strain, deletions were frequently observed in wild-type strain, whilst base substitution and deletion in the mus-52 and mei-3 strains. These mutations resulting from carbon-ion-beam irradiation depend on the mechanism invoked to cope with DSBs. Furthermore, in wild-type cells, these mechanisms likely compete to repair DSBs.